On the functional site of manganese in photosynthetic electron transport system

Normal Euglena chloroplasts contained 1 atom of Mn per 47±8chlorophyll molecules. The manganese content of chloroplastswas decreased by heat treatment. After complete removal of manganeseby incubation at 45°C for 5 min, Hill activity with DPIPas electron acceptor was abolished, but the activity of DPIPphotoreduction with diphenylcarbazide as electron donor wasunaffected. Hill activity was inactivated by incubating Euglena chloroplastsat alkaline pH. The presence of a high concentration of Trisduring incubation of chloroplasts at an alkaline pH had no additionaleffect on the activity drop. Donor-supported DPIP photoreduction in heated Euglena chloroplasts,as well as the normal Hill reaction in untreated chloroplasts,was inhibited by DCMU, HOQNO and ioxynil which block electrontransport at the reducing side of system II. These reactionswere also inhibited by another group of inhibitors; CCCP, salicylaldoxime,antimycin A and azide, which block electron transport at a sitebetween the electron carriers, Y₁ and Y₂ located on the oxidizingside of system II. Possible sites of inhibition by heat treatment and by inhibitorsand sites for entry of electrons from artificial electron donorsin the photosynthetic electron transport chain, especially inrelation to the functional site of endogenous manganese in chloroplasts,were proposed. (Received October 30, 1971; )     

Two modes of inhibition by 2-heptyl-4-hydroxyquinoline- N-oxide of electron transport associated with photosystem 2 in spinach chloroplasts

2-Heptyl-4-hydroxyquinoline-N-oxide (HOQNO) exerted two modesof inhibition on the Hill reaction in spinach chloroplasts.The first mode of inhibition by HOQNO, which developed rapidlyand reversibly on addition of the inhibitor to chloroplasts,was observed in the Hill reaction with various electron acceptorsand in photoreduction of methyl viologen with ascorbate as electrondonor, but was not observed in methyl viologen photoreductionsupported by reduced DPIP. Fifty percent inhibition of the Hillreaction was obtained with HOQNO at a low concentration of theorder of 10^–6 M. Besides the fast-developing inhibition, HOQNO caused a progressivedecrease in the Hill activity during the reaction when ferrricyanidewas used as electron acceptor. Similar time-dependent inactivationoccurred when chloroplasts were incubated with HOQNO and ferricyanidein the dark. Chloroplasts, thus inactivated, showed loweredactivity for ascorbate-supported methyl viologen photoreductionbut had unchanged activity for photoreduction with reduced DPIPas electron donor. Progress of the time-dependent inhibitionwas accelerated by increasing the concentration of HOQNO orferricyanide and was suppressed by addition of ferrocyanide.On changing the ratio of ferri- to ferrocyanide in the reactionmedium, fifty percent protection of the Hill activity was obtainedwith a ferri- and ferrocyanide mixture giving the redoxpotentialof 560 mv. A simple kinetic model for the second time-dependentinhibition by HOQNO is presented. ¹ Present address: Department of Botany, Faculty of Science,Toho University, Narashino 275, Chiba, Japan. (Received January 19, 1971; )     

Conservation of electron transport and energy transfer reactions of spinach chloroplasts in glycero

When spinach chloroplasts were suspended in 50% glycerol inthe presence of 5 mM sodium chloride, then stored at –20°Cactivity of the Hill reaction was retained for several monthswith a small loss. Under these conditions photophosphorylationactivity was reduced by half during the month of storage. (Received February 2, 1971; )     

Biophysical studies on subcellular particles V. Effects of temperature on the ferricyanide-Hill reaction, the light-induced pH shift and the light scattering response of isolated spinach chloroplasts

Effects of temperature on the ferricyanide-Hill reaction, thelight-induced pH shift (JpH) and the light scattering response(4LS) of isolated spinach chloroplasts were studied at temperaturesranging from 5 to 60°C. Activities which produce JpH and JLS under actinic illuminationdecreased with a rise in the reaction temperature above 30°Cand disappeared at 50°C. The ferricyanide-Hill reactionat pH 6.0 was stimulated by raising the reaction temperaturefrom 5 to 35°C, was slowed at higher temperatures and wasinactivated at 50°C. 4LS activity at temperatures below20°C depended on the activity of electron transport. With a rise in temperature of a suspension of chloroplasts whichexhibited JLS under actinic illumination, the scattering intensitydecreased. The decrease in scattering intensity was limited,at most, to an extent equal to ALS. The behavior of JLS whichdiffered from that of basal scattering is discussed in connectionwith a mechanism of JLS formation other than the shrinkage-swellingcycle. When activities were measured at 25°C with chloroplastswarmed transiently (2 min) at various temperatures (25–60°C),the dependence of JLS on the wanning temperature was identicalwith that on the reaction temperature, while the activitiesof both ferricyanide reduction and JpH formation were higherthan those measured at the corresponding reaction temperature.This suggested that the mechanism for 4LS formation could beirreversibly altered by a warming treatment and that it wasless stable against heat than the mechanisms of ferricyanidephotoreduction and 4pH formation. That is, transiently warmedchloroplasts showed a similar behavior to alkane-treated chloroplasts.The similarity is discussed in relation to the activity changethrough the possible and common cause of disordering of theorganized structures of lipophilic components in the lamellarmembranes. (Received June 5, 1971; )     

Electrostatic state of the membrane surface and the reactivity between ferricyanide and electron transport components inside chloroplast membranes

The relationship between the electrostatic state of the thylakoidmembrane and the photoreduction rate of ferricyanide were studiedin intact and sonically treated chloroplasts. In sonicated chloroplasts, reduction of ferricyanide by photosystemII required a high concentration of ferricyanide. for full activity,the apparent low affinity of the reduction site for ferricyanidesuggests that permeation of ferricyanide to the site is therate-limiting factor. The affinity was increased by additionof salts or a cationic detergent, but not by anionic or nonionicdetergent at neutral pH. It was also increased by decreasingpH or by adding lipophilic electron mediators such as naphthoquinonesor p-phenylenediamine. In intact chloroplasts similar characteristics of the systemII reducing site were also observed in the presence of dibromothymoquinone,an antagonist of plastoquinone. It is suggested from these results that the site of ferricyanidereduction in system II exists inside the membrane with negativesurface charges, which hinder the access of ferricyanide tothe site by electrostatic repulsion. The charges were probablyscreened by salts or decreased by protonation at low pH. Studywith the fluorescent probe, 8-anilinonaphthalene-1-sulfonate,in sonicated chloroplasts also showed the pH- or salt-inducedchanges and confirmed the interpretation above. From the rate constant for ferricyanide reduction, electricalpotential difference of the membrane surface with respect tothe bulk aqueous phase was estimated by assuming potential-inducedchanges in the ferricyanide concentration at the membrane surface.A potential of –25––30 mV and a surface densityof 1 negative charge per 12–22 chlorophylls were estimatedat pH 7.5 under low salt conditions. This potential was reducedby salt addition and changed the sign to positive at low pH(+17 mV at pH 4.9). The effect of the surface potential in photosynthetic energyconversion is discussed. (Received September 5, 1977; )     

Some Factors Affecting the Hill Reaction Activity in Cotton Chloroplasts

A method of plant culture was developed for growing large leaves of glandless cotton on single stems. Chloroplasts isolated from these leaves actively reduced ferricyanide when assayed for the Hill reaction. Hill reaction activity increased 133% when the 0.5 m sucrose isolation medium was replaced with 10% (w/v) polyethylene glycol, both buffered at pH 7.6. The presence of 2 or 5% (w/v) bovine serum albumin in the sucrose buffer did not increase Hill activity. Ferricyanide reduction in the dark occurred in all assays, and the possibility of gossypol as the reductant is discussed. Half-life of the chloroplasts stored in 10% glycerol at -23 C was 23 days. The ammonium ion at 0.01 m enhanced Hill reaction activity up to 171%. Leaves containing chloroplasts with the highest Hill reaction activity were found near the 8th node below the apex. Leaf water potentials less than -28 bars reduced the activity about 50%. Daylight conditions during the winter months in the greenhouse reduced the activity about 30%.     

Fatty acid synthesis by spinach chloroplasts I. Property of fatty acid synthesis from acetate

Intact chloroplasts (about 70% Class I chloroplasts) isolatedfrom spinach leaves incorporated 150 nmoles of [1-¹⁴C] acetateinto fatty acids per mg chlorophyll in 1 hr at pH 8.3, 25°Cand 25,000 lux. On electron and phase-contrast microscopiescombined with hypotonic treatment of chloroplasts, this syntheticactivity was shown to be proportional to the percentage of ClassI chloroplasts in the preparation. Light was necessary for thesynthesis, the activity in the complete reaction mixture inthe dark being only 2% of that in the light. The synthetic activityincreased with increasing intensities of light to reach saturationat 6,000 lux. CoA and ATP were most effective as cofactors,HCO₃^–, HPO₄^2–, Mg^2$ and Mn^2$ were less effective.ATP could be replaced by ADP in the presence of Pi, suggestingpossible supply of ATP by photophosphorylation. Omission ofthe NADPH-generation system and NADH did not affect the synthesis,indicating sufficient provision of endogenous NADPH and NADHin intact chloroplasts under light. Addition of DTE did notcause recovery of the synthetic activity of intact chloroplastsin the dark. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Mechanisms of the acido- and thermo-phily of Cyanidium caldarium Geitler I. Effects of temperature, pH and light intensity on the photosynthetic oxygen evolution of intact and treated cells

Photosynthetic oxygen evolution in an acido- and thermo-philicunicellular alga, Cyanidium caldarium, was measured under variousconditions, using a Clark-type oxygen electrode. 1). Maximum Hill reaction activity with p-benzoquinone as theHill oxidant was obtained at 45°C in a wide pH range from1.0 to 7.0. 2) The pH activity curve showed two peaks at pH3.0 and 7.0. The Hill activity had an optimum at pH 3.0 in cellspreilluminated under strong light (300,000 lux, 30 min, 40°C).Sonication of algal cells abolished the pH 3.0 component ofthe Hill reaction producing an activity maximum at pH 7.0. 3)Endogenous O₂ evolution in the absence of the Hill oxidant,which lasted for several minutes after illumination, had a maximumat pH 7.0. 4) This endogenous O₂ evolution was abolished bysonication. 5) KCN inhibited endogenous O₂ evolution, but notthe Hill reaction in the presence of p-benzoquinone. (Received August 19, 1974; )     

Effect of {alpha}-Tomatine on the Integrity and Biochemical Activities of Isolated Plant Cell Organelles

Respiration in isolated mung bean shoot mitochondria was notaffected by either of the tomatine treatments (10^–3 M,pH 5, and 10^–5 M, pH 8) but was reduced in the excisedshoots by both treatments, although only at the higher of thetwo temperatures (5 °C and 25 °C). Inhibition was gradualand took at least 2 h. Tomatine treatment of excised shootsalso resulted in an increased leakage of K+. PS II activityin isolated spinach leaf chloroplasts was reduced only by thehigh pH tomatine treatment at 25 °C. Again, about 2 h treatmentwas required before significant effects were observed but thealkaloid did not cause disintegration of the chloroplast asmeasured by pigment release. Disruption of lysosomes isolatedfrom cauliflower inflorescence tissue and release of acid phosphatasewas enhanced by tomatine. Initially only 10^–5 M tomatineat pH 8 was effective but, later, effects could only be obtainedwith 10^–3 M alkaloid at pH 5. The differential susceptibilityof these organelles to tomatine is discussed in relation tomembrane structure and to the mode of toxicity of the alkaloid.     

Removal of Mn from spinach chloroplasts by sodium cyanide and the binding of Mn2+ to Mn-depleted chloroplasts.

Manganese and copper were released from spinach chloroplasts by NaCN-treatment, though iron was not affected. The Hill reaction activity was also inhibited by this treatment, but was partially recovered by the addition of either Mn2+ or Cu2+, but not of Fe3+. The interaction of Mn2+ with manganese-depleted chloroplasts by NaCN-treatment was studied using 54Mn2+. A Scatchard plot shows the high and low affinity binding sites of Mn2+ on NaCN-treated chloroplast membrane; high affinity binding being specific for NaCN-treated chloroplast with a binding constant, KH, of 1.9 X 10(5) M-1, and a maximum binding number, NH, of 0.0016 g-atom per mole of chlorophyll. The low binding site was also found on untreated chloroplasts; its binding constant, KL, being 1.2 X 10(4) M-1, and its maximum binding number, NL, of 0.0112 g-atom per mole oc chlorophyll at pH 8.2 NH was proportional to the degree of the removal of Mn by NaCN-treatment and was constant at pH 4--9. NL markedly increased at a high pH with a midpoint of pH 7.9 indicating the exposure of a new, similar binding site. Light illumination partially inhibited the binding of Mn2+. Within 1 min in the dark the binding reaction reached equilibrium in the absence of pyrophosphate, however, 20 min were required to transform into pyrophosphate-resistant form. The pH dependence of the binding of Mn2+ with pKa 7.2 and the ineffectiveness of p-chloromercuribenzoate suggest the possible ligand of Mn2+ is the imidazole nitrogen of the histidine residue.     

Studies on photophosphorylation II. Uncoupling of chloroplasts by anions at low temperatures and at acidic pH values

Photophosphorylation of spinach chloroplasts was uncoupled bypreincubation at 0°C in the presence of a neutral salt atpH 6.0 to 6.5 ("cold-anion uncoupling"). Preincubation at 20°Ccaused some depression in both photophosphorylation and theHill reaction, but the efficiency of photophosphorylation wasnot depressed much. Low pH values accelerated uncoupling. Theeffectiveness of anions tested as sodium salts in inducing uncouplingwas of the order: SCN->>NO₃^–>Cl^–>SO₄^2–There was little difference in effectiveness among monovalentcations; LiCl, NaCl, KCl, RbCl and CsCl. 10^–4M ATP orADP largely protected chloroplasts from cold-anion uncoupling.Addition of EDTA-extract or dicyclohexylcarbodiimide to uncoupledchloroplasts partially restored photophosphorylation. Theseobservations suggest that inactivation of chloroplast ATPaseis one cause of cold-anion uncoupling. At low light intensities, the time lag and the depression ofthe efficiency of photophosphorylation were more pronouncedin cold-anion uncoupled chloroplasts than in the control chloroplasts. (Received February 15, 1972; )     

Photosynthetic Properties of Green Barley Leaves after Treatment with Pyridazinone Herbicides--Comparison with the Effects of Diuron

Photosynthetic characteristics of detached green barley leavesafter 72 h of treatment with 0·2 mol m^–3 of thepyridazinone herbicides SAN 6706, SAN 9785 and SAN 9789 werestudied. For comparison, the effects of 0·01 mol m^–3diuron were also investigated. Pyridazinone herbicides causedonly a slight reduction of the total carotene content of thebarley leaves. The total chlorophyll content, as well as thelinolenic to linoleic acid ratio of chloroplast glycerolipids,however, remained unchanged. Diuron treatment caused total inhibitionof electron transport, as revealed by fast fluorescence inductionof leaves and the Hill reaction activity of chloroplasts. The¹⁴CO₂-nxation by the leaves and the light-induced fluorescencequenching were also completely inhibited in vivo by diuron.Pyridazinone herbicides left 20–40% of the ¹⁴CO₂-fixationfound in the control, in spite of the fact that their fast fluorescenceinduction tracings showed inhibition in the electron transport.Chloroplasts isolated from the leaves treated with pyridazinoneswere found to be highly active in mediating the ferricyanide-dependentHill reaction. In order to test the ability of pyridazinonesto inhibit photosynthetic electron transport in vivo, their‘prompt’ effect on fluorescence was also investigated.It is concluded that pyridazinone herbicides can readily andrapidly enter the chloroplasts and inhibit the photosyntheticelectron transport in vivo. The differences between the long-termeffects of pyridazinones and those of diuron suggest differencesin the inhibitory effectiveness on the various photosyntheticparameters between the two herbicide groups. It is suggestedthat pyridazinones can leave the chloroplasts during isolationowing to the loose binding onto the thylakoid membranes. Key words: Pyridazinone herbicides, electron transport, fluorescence induction     

Coupling of Proton Fluxes in the Polar Leaves of Potamogeton lucens L

An attempt has been made to quantify the light-induced H⁺ effluxand influx observed in polar leaves of Potamogeton lucens.Theseproton fluxes are spatially separated. The H⁺ efflux, mediatedby a plasmalemma bound H⁺ –ATPase, occurs across theplasmamembrane at the morphological lower epidermis and is accompaniedby an H⁺ influx (or OH^– efflux) at the upper side oftheleaf. As a result, these leaves exhibit a remarkable pH–polarityin the light. The pH near the lower epidermis may drop to avalueas low as 3.5, while a pH of about 10.5 can be observed at theupper epidermis. Obviously this phenomenon requires theco–ordinationof transport processes in the different cell layers of the leaftissue. These observations led to quantitative studies oftherelation between the H⁺ fluxes at either plasmalemma. Thesefluxes were calculated from the pH values recorded at twodistancesfrom the leaf surface. Although the H⁺ influx always exceededthe efflux, a coupling between the transport processesacrosseither plasma membrane became evident from the time–coursesof the two fluxes. Key words: Potamogeton lucens, proton flux, flux coupling, pH–;polarity     

Release of Free Fatty Acids and Loss of Hill Activity by Aging Spinach Chloroplasts

The free fatty acid content of spinach chloroplasts, isolated at pH 5.8 to 8.0, has been found to vary between 3.1 and 5.5% of the total chloroplast fatty acids. When chloroplasts were incubated at room temperature for 2 hours, the free fatty acids increased by 42% and the Hill activity decreased by 70%. After 2 hours of incubation at 37 degrees , the free fatty acids increased about 3-fold and the Hill activity decreased to almost 0. The addition of crystalline bovine serum albumin largely prevented the loss of Hill activity at room temperature and at 5 degrees , but had little effect during incubation at 37 degrees . Both the release of free fatty acids and the loss of Hill activity were pH dependent. The losses were the least during incubation at pH 5.8 and the greatest during incubation at pH 8.0. The major free fatty acids released at pH 5.8 were saturated, while those released at pH 7.0 or 8.0 were mainly the unsaturated acids, alpha-linolenic acid and hexadecatrienoic acid.     

Effects of chloride ion on HILL reactions in Euglena chloroplasts

Effects of Cl^– and other anions on the rate of HILL reactionin Euglena chloroplasts were investigated. Cl^– acceleratedthe reaction rate with ferricyanide as HILL oxidant; Br^–,F^– and I^– were also effective; NO₃^–, PO₄^2–and SO₄^2– were less effective. Divalent cations, Ca²⁺and Mg²⁺, were also highly effective. The promoting effectsof these ions were highly dependent on pH and the nature andconcentration of the HILL oxidant used. Accelerating effectsof the ion increased with decreasing concentrations of ferricyanide.Generally, the stimulating effect of Cl^– was much moremarked at pH 7–7.5, with little effect at pH 5. Thus,the pH-activity relationship in the HILL reaction is more orless markedly modified by addition of ions. Cl^–, and other anions, accelerated the reaction by affectingonly the dark rate-limiting portion of the HILL reaction; thelight reaction constant remained uninfluenced. We inferred thatsome reaction step, at which ferricyanide receives electronfrom photosystem 2, is accelerated by Cl^– and other ions.Cl^– effects were rather small, or undetectable, with DPIPor p-benzoquinone as oxidants. (Received January 8, 1970; )     

Comparative study of the action of ultraviolet-C and ultraviolet- B radiation on photosynthetic electron transport

The effect of ultraviolet-C (UV-C, mainly 254 nm radiation) and ultraviolet-B (UV-B, 290-320 nm) radiation on the photosynthetic electron transport reactions has been investigated. The rates of Hill activity mediated by ferricyanide and dichlorodimethoxy-p-benzoquinone (DCDMQ) were differently sensitive to UV-C but equally inhibited by UV-B. Replacement of water with diphenylcarbazide was ineffective in restoring the activity of dichlorophenol indophenol (DCPIP) Hill reaction in UV-B treated chloroplasts, but had significant effect in UV-C treated chloroplasts.
Photobleaching of carotenoids in the presence of carbonyl cyanide-m-chlorophenyl-hydrazone, an indicator of the photochemical reaction associated with the reaction centre of photosystem II, was suppressed and is paralleled by the changes in Hill activity only in UV-B-treated chloroplasts. Carotenoid photobleaching occurred even in UV-C treated chloroplasts showing no measurable Hill activity. UV-C and UV-B irradiation diminished variable fluorescence. With UV-B treated, but not with UV-C treated chloroplasts, an increase in the fluorescence yield was observed upon the addition of 3-(3,4-dichIorophenyl)-l,l-dimethylurea (DCMU) and/or Na dithionite.
Photosystem I activity was found to be unaffected by both UV-C and UV-B radiation at the fluences tested. Kinetics of P700 photooxidation and dark reversal in UV treated chloroplasts indicate that only the electron flow from photosystem II to photosystem I is impaired. It is concluded that while UV-B radiation inactivates specifically the photosystem II reaction centre, UV-C radiation acts at plastoquinone, the quencher Q, and the water oxidizing enzyme system.     

Efficient purification and molecular properties of spinach chloroplast fructose 1,6-bisphosphatase.

A relatively straightforward procedure has been developed for the purification of chloroplast fructose bisphosphatase from spinach leaves to apparent homogeneity and with 80% yield. The molecular weight of the enzyme was about 160 000. Chloroplast fructosebisphosphatase consists of four possibly identical subunits and, at pH 8.8, EASILY DISSOCIATES INTO EQUAL HALVES WITH LOWered activity. Sigmoid saturation curves with Hill coefficients between 3.0 and 3.7 were obtained for fructose 1,6-bisphosphate and Mg2+. Incubation of the enzyme with 20 mM dithiothreitol slowly altered the response to pH from no activity measured at pH 7.5 and full activity at pH 8.8 to equal activity at each of these pH values; at the same time the number of freely available sulphydryl groups increased from four to twelve per molecule. These properties are considered in the context of the observed activation of this enzyme following illumination of chloroplasts.     

The Hill reaction and oxygen uptake in isolated pine chloroplasts

1. The Hill reaction and oxygen uptake in chloroplasts preparedfrom pine leaves were studied. Pine chloroplasts active forthe Hill reaction were isolated from mature leaves in the presenceof 25% PEG in the isolation buffer. Time courses of the Hillreaction in chloroplasts isolated from leaves at different seasonsdiffered. In chloroplasts isolated in the autumn, Hill activitydecreased rapidly with illumination time. This rapid decreaseof Hill activity was inferred to result from concomitant productionof some inhibitory substance(s) during the Hill reaction.
2. Theprotective effect of PEG on inactivation by aging of pinechloroplastswas found. In the presence of PEG, chloroplastswere stabilizedand Hill activity was maintained even afterstorage for 26 hr;whereas, in the absence of PEG inactivationby aging proceededrapidly and oxygen uptake occurred after20 hr.
3. Chloroplastsisolated without PEG had no ability of the Hillreaction; but,inversely showed pronounced oxygen uptake. Oxygenuptake wasalso observed in aged or DCMU-inhibited chloroplasts.The presenceof benzoquinone strongly suppressed oxygen uptake.

¹ Present address: Laboratory of Biology and Chemistry, Universityof Naval Architecture of Nagasaki, Nagasaki, Japan. (Received January 27, 1971; )     

The Effect of Heat Shock on the Capacity of Wheat Plants to Restore Their Photosynthetic Electron Transport after Photoinhibition or Repeated Heating

The shoots of 16-day-old spring wheat plants (Triticum aestivumL., cv. Albidum 29) were subjected to heat shock (HS) at 40, 41, or 43°C for 10 min. The activity of the Hill reaction in chloroplasts isolated immediately after HS was 83, 61, and 30% of the initial value, respectively. The activity of the Hill reaction was also estimated after plant return to the initial growth conditions for one day. It was completely restored after heating at 40°C and achieved 82 and 30–33% of the initial level after heating at 41 and 43°C, respectively. Thereafter, the shoots were heated repeatedly at 42, 43, or 43.5°C for 10 min, and the activity of the Hill reaction was measured immediately or one day after this heating. Immediately after the second heating, the activity decreased again as compared to its value before heating. The percent of inhibition of the Hill reaction was similar in the control plants not subjected to preliminary HS and HS-treated plants independently of the temperature used. However, after one-day growth under normal conditions, the activity of the Hill reaction was restored almost completely in HS-treated plants but not more than by 10% in the control plants. The conclusion is that different mechanisms underlie the development of the tolerance to HS and recovery. Some plants were tested for the effect of HS (40°C) on their tolerance to photoinhibition. The degree of the Hill reaction inhibition after plant exposure to the light of 65–75 klx for 3 h was essentially similar in detached leaves from the HS-treated and unheated plants and comprised about 40% of the activity before light stress. After the leaves were returned to the low-light conditions for 3 h, the Hill reaction was restored and attained about 75% of that before photoinhibition in both HS-treated and untreated plants. The lack of the HS-induced stimulation of the Hill reaction recovery after photoinhibition is evidently related to the fact that heating and excess light damage different sites of photosystem II, which implies the different pathways for the recovery of its functional activity.     

Light-Dependent Uptake of Pyruvate by Mesophyll Chloroplasts of a C4 Plant, Panicum miliaceum L.

Light-dependent active uptake of pyruvate was reported in mesophyllchloroplasts of a C₄ plant, Panicum miliaceum [Ohnishi and Kanai(1987) Plant Cell Physiol. 28: 1]. The present study tried toclarify the energy source of this active uptake. Preilluminationof the mesophyll chloroplasts increased over tenfold their pyruvateuptake in the light and dark. This indicates that light itselfis not essential for the enhancement. The pyruvate uptake capacity(the initial uptake rate) of the mesophyll chloroplasts increasedon illumination and reached a steady-state level after a fewminutes; this rise was faster under higher light intensities.When the chloroplasts were returned to darkness, the uptakecapacity decayed with a half-life of about 1 min; this was independentof the light intensity of preillumination. Illumination of thechloroplasts also increased the stromal pH from about 7 to 8and the stromal ATP level from about 5 to 15–25 nmol.(mg chl)^–1. The change of the former during dark-to-lightand light-to-dark transitions occurred within 2 to 5 min, whilethe change of the latter took place much faster within 1 min.The steady-state levels of the pyruvate uptake capacity andstromal pH were saturated at a light intensity of 3 µE.m^–2.s^–1,while the ATP level increased with a further increase in thelight intensity. The former two parameters also showed similarsensitivity to the inhibition by carbonylcyanide-m-chlorophenylhydrazone,while a higher concentration of the inhibitor was needed toreduce the ATP level. Nitrite at 4 mM inhibited the light-dependentpyruvate uptake and stromal alkalization but had little effecton the stromal ATP level, while 2 mM arsenate decreased thestromal ATP without significant effects on pyruvate uptake andstromal pH. The good correlation of pyruvate uptake and stromalpH suggests that the active pyruvate uptake by the mesophyllchloroplasts is primarily driven by the pH gradient across theenvelope. (Received August 15, 1986; Accepted December 8, 1986)